JPS5823380B2 - Formilkino Datsurihou - Google Patents

Description

Detailed Description of the Invention The present invention provides a method for eliminating formyl groups, more specifically, a method for eliminating formyl groups more efficiently and preferentially than from compounds containing amino groups and ester groups protected by formyl groups. Regarding the method.

The present invention provides particularly effective means for peptide synthesis, and includes ester groups such as N-formyl amino acid esters, N-formyl peptide esters, and especially N-formyl asbartyl peftide esters. In addition, it exhibits a superior effect in eliminating formyl groups compared to compounds with free carboxyl groups.

Hereinafter, the case where the method of the present invention is applied to N-formyl amino acid esters and N-formyl peptide esters will be mainly explained, but the present invention is not limited thereto, and the present invention is not limited to these. The present invention is generally applicable to compounds containing N-formyl ester groups and ester groups (hereinafter referred to as N-formyl ester compounds).

Generally, benzyloxycarbonyl groups and t-butyloxycarbonyl groups are used as protecting groups for amino groups, especially in peptide synthesis, but these protecting groups require dangerous phosgene gas to be introduced. It is unsuitable for factory scale production.

On the other hand, the formyl group used in the present invention is an advantageous method for industrial production from the viewpoint of ease of introduction and cost, but as described below, there are problems in its removal, so it is hardly used. This is the current situation.

Conventionally, it has been recommended that the formyl group be removed by acidic hydrolysis, acidic alcoholysis, catalytic reduction, or the like.

However, acidic hydrolysis carries the risk of hydrolysis of ester groups and cleavage of peptide bonds, while catalytic reduction does not proceed unless special conditions are met.

In addition, a method using hydrazine (D, La
francier.

Bull, Chem, Soc, France, 196
5,3668) and the method using hydroxylamine or its weak acid salt (Rolf Geiker, Japanese Patent Publication No. 45-289
66 specification), but even if these methods are applied to N-formyl compounds containing ester groups, in the former case the ester group changes to hydrazide, and in the latter case the ester group changes to hydroxamic acid. or diketopiperazine derivatives in the case of dipeptide esters, making it impossible to achieve the desired deformylation.

In order to effectively utilize formyl group as an effective protecting group for amino groups, especially as an amine protecting group for peptide ester synthesis, the present inventors investigated various methods for removing the formyl group, which is an obstacle to this, and found that hydroxylamine strong acid When the salt is applied to an N-formyl ester compound, surprisingly, the formyl group can be selectively eliminated without causing the various side reactions described above, and furthermore, it is possible to remove the formyl group selectively without causing the various side reactions described above. Or, it has been found that even when a weak acid salt of hydroxylamine is used in combination, results equivalent to or better than when a strong acid salt is used alone can be obtained,
The invention has been completed.

The strong acids constituting the strong hydroxylamine salt used in the present invention include mineral acids such as hydrochloric acid and sulfuric acid, monomethyl sulfuric acid,
A strong organic acid such as benzenesulfonic acid or trifluoroacetic acid with a first dissociation constant of 1.0X10' at 25°C.
A strong acid that is above is used.

Among these strong acids, mineral acids, particularly salts of hydrochloric acid, sulfuric acid, etc., are most preferably used because they are easily available industrially and are easy to handle.

These strong acid salts are usually introduced into the reaction system in the form of salts,
Of course, free hydroxylamine or a weak acid salt thereof and a strong acid may be individually introduced into the reaction system to form a strong acid salt before use.

On the other hand, the esters constituting the entire N-formyl ester compound as a raw material are esters with primary and secondary alcohols, and there are no other limitations.

Typical examples include esters with methyl alcohol, ethyl alcohol, inflovir alcohol, benzyl alcohol, etc., which are commonly used in peptide synthesis.

In addition, carboxylic acids such as acetic acid and propionic acid are typical examples of weak acids constituting the weak acid salt of hydroxylamine.

A feature of the present invention is that the elimination reaction proceeds easily and the target product can be obtained in high yield.

That is, the reaction proceeds simply by adding the N-formyl ester compound to a solution of a strong hydroxylamine salt, or a solution of the strong salt and free hydroxylamine and/or the weak salt.

In addition, compounds constituting the N-formyl ester compound,
For example, there are no restrictions on the type of amino acids such as N-formyl amino acid esters and N-formyl peptide esters, which include carboxyl groups, amide groups, amino groups, thiol groups, etc. in addition to peptide groups and ester groups. The reaction proceeds without any problem.

Therefore, it is particularly suitable for the deformylation reaction of N-formyl asbartyl dipeptedoester, which has a carboxyl group and an ester group, from which it has been difficult to remove the formyl group, and has attracted attention as a sweetening agent. It also provides an effective means for the synthesis of aspartyl dipeptide esters.

When an N-formyl ester compound, a strong hydroxylamine salt, free hydroxylamine, and/or a weak salt thereof are present, the solvent used in the present invention should be inert to the amines and the product. There is no particular restriction as long as it is water, alcohols (methanol, ethanol, n-furopanol, isopropanol, n-butanol, glycerol, propylene glycol) that have high solubility for both reactants and products. Such)
, or a mixed solvent of water and alcohol.

There is no particular restriction on the reaction temperature, and the reaction proceeds quickly at high temperatures as in normal chemical reactions, but since there is a risk of racemization or change to diketopiperazine derivatives in the case of dipeptide esters, it is preferable to °C to 80 °C is selected.

Furthermore, the reaction completion time depends on the reaction temperature as mentioned above,
For example, the heating time is 1 to 4 hours at 70°C, 5 to 12 hours at 50°C, and about 2 to 4 days at around 25°C.

There is no particular restriction on the amount of the strong hydroxylamine salt to be used, but it is usually preferred to use it in an amount equal to or more than the equivalent molar amount, particularly 1.5 to 7 times the molar amount of the N-formyl ester compound.

In addition, when free hydroxylamine and/or a weak acid salt thereof are present together, the above-mentioned amount may be used as the sum of the amine and the strong acid salt.

The amount of the free form and the weak acid salt to be used is preferably 30 mol% or less of the strong hydroxylamine salt, and if the amine is present in a large amount, the free hydroxyl amino acid or the weak acid salt will be used alone. Similarly, there is a risk that esters will change to hydroxamic acid, or dipeptide esters will change to diketopiperazine derivatives.

Free hydroxylamine or its weak acid salt may be introduced directly into the reaction system using free hydroxylamine, but since it is unstable in air, it is usually introduced after introducing a strong hydroxylamine salt into the reaction system. This is carried out by neutralizing a part of it with a compound more basic than hydroxylamine, such as potassium hydroxide, sodium acetate, etc., to form free hydroxylamine or its weak acid salt.

Isolation of the desired deformylated product can be easily carried out according to a conventional method after the completion of the reaction.

That is, after reacting for a predetermined period of time, if necessary, the reaction solution is stored at a low temperature and filtered to obtain pure deformylated products such as amino acid esters and peptide esters.

When the target product has high solubility, it is sufficient to concentrate the product and then add a solvent in which the target product has low solubility for crystallization.

As is clear from the above explanation, according to the present invention, formyl groups can be selectively eliminated from various N-formyl ester compounds without causing side reactions such as changes in ester groups, which is industrially extremely advantageous. .

A more detailed explanation will be given below using Examples and Comparative Examples.

Unless otherwise specified, the yield in each Example is the molar yield based on the N-formyl ester compound used, and was determined as a result of quantitative analysis as follows.

That is, the reaction solution was appropriately diluted, a certain amount was taken, filter paper electrophoresis was performed, and ninhydrin-cadmium reagent (Z, phys
iol, Ch-em, 309, 219 (1957))
After color development, a portion corresponding to the half-formed deformylated product was cut out and extracted with methanol, and the yield was determined by measuring and quantifying the absorbance of the extract at 510 mμ.

Example I 9.6 g (30 mm!J mol) of N-formyl-α-L-aspartyl-L-phenylalanine methyl ester and 10.4 g of hydroxylamine hydrochloride. (150 mmol) was added to 36 ml of 90% aqueous methanol solution, and after heating and stirring at 70° C. for 3 hours, a portion of the reaction solution was taken out and subjected to quantitative analysis.

The yield of α-L-aspartyl-L-phenylalanine methyl ester in the reaction solution was 86%.

After quantitative analysis, the reaction solution was concentrated, and the residue was added with 40 m of 3N-hydrochloric acid.
1 was added and stored in the refrigerator overnight.

8.5 g of precipitated α-L-aspartyl-L-phenylalanine methyl ester hydrochloride was collected by filtration.

The crystals were dissolved in 50 mA of water, adjusted to pH 4.8 using carbonic acid, and stored overnight in the refrigerator to release free α-
6.8 g of L-aspartyl-L-phenylalanine methyl ester was obtained.

Yield 75% [α)j'=-+-32,2° (C=1,
Example 2 N-formyl-L-aspartic acid-α-methyl ester (1.75 i10 mmol) and hydroxylamine arsenate (3.5 g (50 mmol)) were added to 90% aqueous methanol solution (107 Ill) After stirring at 70°C for 4 hours, quantitative analysis was performed.

The yield of L-aspartic acid-α-methyl ester in the reaction solution was 85%.

Example 3 2.07 g (10 mmol) of N-formyl-L-phenylalanine methyl nisol and 3.5 g (50 mmol) of hydroxylamine hydrochloride were added to 127 g (50 mmol) of methanol.
7+l and further add 0.82.9(1
0 mmol) was added to neutralize a portion of hydroxylamine hydrochloride to form an acetate salt, and after stirring at 70°C for 1.5 hours, quantitative analysis was performed.

The yield of L-phenylalanine methyl ester in the reaction solution was 80%.

Example 4 2.3 g (10 mmol) of N-formyl-glycyl-L-leucine methyl ester and human hydrochloride.

3.5 g (50 mmol) of xylamine in 1 part of methanol
Dissolve in 2 ml and add 0.414 (5 mmol) of sodium acetate to neutralize a portion of the hydrochloride to form acetate,
Quantitative analysis was performed after stirring at 70°C for 2 hours.

The yield of glycyl-L-leucine methyl ester in the reaction solution was 88%.

Example 5 1.6 g (5:!J mol) of N-formyl-α-L-aspartyl-L-phenylalanine methyl ester and 1.71' (25 mmol) of hydroxylamine hydrochloride were added to 8 ml of methanol and stirred at 50°C for 10 hours. Quantitative analysis was then carried out.

The yield of α-L-aspartyl-L-phenylalanine methyl ester in the reaction solution was 83%.

Example 6 N-formyl-α-L-aspartyl-L-phenylalanine ethyl ester 1. '1 (5-: rimole) and 1.7 g (10 mmol) of hydroxylamine sulfate at 75
% Ingrofluor aqueous solution 107,721 and after stirring for 4 hours quantitative analysis was performed.

The yield of α-L-aspartyl-L-phenylalanine ether ester in the reaction solution was 74%.

Example 7 N-formyl-α-L-aspartyl-L-f rosin ethyl ester 1. 1 (5 mm! J mol) and 1.4 g (20:: mol) of hydroxylamine hydrochloride were added to 8 ml of a 90% aqueous methanol solution, and after stirring at 70° C. for 3 hours, quantitative analysis was performed.

The yield of α-L-aspartyl-L-tyrosine ethyl ester in the reaction solution was 83%.

Example 8 1.4 g of N-formyl-L-aspartic anhydride (1
0 mmol) and L-phenylalanine methyl ester 1
．． ! 1 (10 mmol) was condensed in ethyl acetate 207111.

The reaction solution was concentrated, and the residue contained 30 ml of methanol, 3.5.9 ml of hydroxylamine hydrochloride (50 mmol), and 0.56 ml of potassium hydroxide. ? (10 mm! J mol) was added to neutralize a portion of hydroxylamine hydrochloride, and after stirring at 70°C for 2 hours, quantitative analysis was performed.

The yield of α-L-aspartyl-L-phenylalanine methyl ester in the reaction solution based on the used N-formyl-L-aspartic anhydride was 70%.

The yield of β-L-7 spartyl-L-phenylalanine methyl ester based on the anhydride was 21%.

Comparative Example 1 1.6 g (5 mmol) of N-formyl-α-L-aspartyl-L-phenylalanine methyl ester and 1.74.9 (25 mmol) of hydroxylamine hydrochloride were dissolved in 10 ml of methanol, and further 2.0 g of sodium acetate was added.
Hydroxylamine hydrochloride was neutralized by adding 5 g (25 mm!J mol) and acetate was added.

The above solution was stirred at 70°C for 1.5 hours.

When the reaction solution was analyzed by thin layer chromatography,
Raw material N-formyl-α-L-aspartyl-L-phenylalanine methyl ester and deformylated product Riruα-
L-aspartyl-L-phenylalanine methyl ester could not be detected.

Comparative Example 2 1.6.9 (5 mmol) of N-formyl-α-L-aspartyl-L-phenylalanine methyl ester and 1.749 (25 mmol) of hydroxylamine hydrochloride were dissolved in 10 ml of methanol, and 4.9 ml of potassium hydroxide was dissolved.
F (25 mm!J mol) was added to neutralize the hydroxylamine hydrochloride.

The above solution was stirred at 70°C for 1.5 hours.

When the reaction solution was analyzed by thin layer chromatography,
Raw material N-formyl-α-I, -7 spartyl-L-phenylalanine methyl ester and deformylated product α
-L-aspartyl-L-phenylalanine methyl ester was not detected.

Claims (1)

[Claims] 1. A method for eliminating formyl groups, which comprises bringing a compound containing a formyl group-protected amine group and ester group into contact with a strong hydroxylamine salt.